Journal: Pharmaceutics

Article Title: Nucleolin-Targeting AS1411 Aptamer-Conjugated Nanospheres for Targeted Treatment of Glioblastoma

doi: 10.3390/pharmaceutics16040566

Figure Lengend Snippet: Nucleolin mRNA and protein expression analysis and flow cytometry and confocal microscopy images of non-target NHA cells and target U87 and U251 glioblastoma cells treated with nanosphere and Apt-Nanosphere. ( A ) Western blot analysis of NCL mRNA expression level of NHA, U87, and U251 cells; β-actin was used as an internal control. ( B ) Real-time RT-PCR analysis of NCL mRNA expression level of NHA, U87, and U251 cells; GAPDH was used as an internal control. Selective binding of AS1411 Apt-Nanosphere to target the U87 ( C ) and 251 glioblastoma cells ( D ) and non-target NHA cells ( E ). ( F ) A comparison of the binding affinity of the FAM-labeled nanospheres and 1411 aptamer-conjugated FAM-labeled nanospheres in the target U87 and 251 glioblastoma cells, as well as the non-target NHA cells. The scale bar for all images is 20 µm. ( G ) The mean fluorescence intensity values were calculated using the ZEN lite software. The values are expressed as mean ± SEM: ** p < 0.01 and * p < 0.05 ( n = 6). Relative quantification was performed using the comparative Ct method (2 −ΔΔCt ). The values are expressed as mean ± SEM: ** p < 0.01 and * p < 0.05 ( n = 4).

Article Snippet: After DNase treatment with RQ1 RNase-free DNase (Promega, Mannheim, Germany), 1 µg of total RNA was used for first-strand cDNA synthesis with oligo-dT primers using the ProtoScript M-MuLV Taq RT-PCR system (New England Biolabs, MA, USA).

Techniques: Expressing, Flow Cytometry, Confocal Microscopy, Western Blot, Quantitative RT-PCR, Binding Assay, Comparison, Labeling, Fluorescence, Software

Journal: The Journal of Cell Biology

Article Title: Myosin VI small insert isoform maintains exocytosis by tethering secretory granules to the cortical actin

doi: 10.1083/jcb.201204092

Figure Lengend Snippet: Myosin VI SI potentiates evoked exocytosis. (A) RT-PCR analysis of myosin VI isoform expression. β-Actin primers were used as a control. (B) Cartoons of GFP-tagged myosin VI constructs. The motor domain (purple) contains the actin- and ATP-binding sites, the reverse gear (761–814 aa, yellow) determines the unique directionality of myosin VI movement along actin, and the tail domain (orange) in the myosin VI SI (1,262 aa), but not the myosin VI NI (1,253 aa), contains a 9-aa insert. GFP-MyoVI-tail constructs lack the motor domain and are unable to interact with F-actin. (C) GFP-MyoVI protein expression in PC12 cells was evaluated by Western blotting using an anti–myosin VI antibody. Arrows indicate GFP-MyoVI-full proteins (175 kD) and GFP-MyoVI-tail proteins (85 kD). (D) NPY-hPLAP release was evaluated using a double-stimulation protocol in PC12 cells expressing GFP (control), GFP-MyoVI-SIfull, or GFP-MyoVI-NIfull. Released NPY-hPLAP is expressed as a percentage of total NPY-hPLAP ( n = 3). (E) NPY-hPLAP release was measured in wild-type (wt) and myosin VI stable knockdown (KD) PC12 cells overexpressing GFP (control), GFP-MyoVI-SIfull (+GFP-MyoVI-SIfull), or GFP-MyoVI-NIfull (+GFP-MyoVI-NIfull). Released NPY-hPLAP is expressed as a percentage of wild-type PC12 cells ( n = 3). Error bars are means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Poly(A) + RNA was reverse transcribed, and PCR products were produced using the RT-PCR kit (ProtoScript M-MuLV Taq; New England Biolabs, Inc.).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Construct, Binding Assay, Western Blot